Community‐level plant palatability increases with elevation as insect herbivore abundance declines

Community-level plant palatability increases with

elevation as insect herbivore abundance declines

Patrice Descombes

*

, Jeremy Marchon

, Jean-Nicolas Pradervand

, Julia Bilat

,

Antoine Guisan

, Sergio Rasmann

and Lo

€ıc Pellissier

1

_{Unit of Ecology and Evolution, University of Fribourg, Ch. du Musee 10, CH-1700 Fribourg, Switzerland;}

Landscape

Ecology, Institute of Terrestrial Ecosystems, ETH Z€urich, CH-8092 Z€urich, Switzerland;

Swiss Federal Research

Institute WSL, CH-8903 Birmensdorf, Switzerland;

Department of Ecology and Evolution, University of Lausanne,

Biophore, CH-1015 Lausanne, Switzerland;

Institute of Earth Surface Dynamics, University of Lausanne, Geopolis,

CH-1015 Lausanne, Switzerland; and

Laboratory of Functional Ecology, Institute of Biology, University of Neuch^atel,

CH-2000 Neuch^atel, Switzerland

Summary

1. Plants protect themselves against herbivore attacks through a myriad of physical structures and

toxic secondary metabolites. Together with abiotic factors, herbivores are expected to modulate plant

defence strategies within plant assemblages. Because the abundance of insect herbivore decreases in

colder environments, the palatability of plants in communities at higher elevation should shift in

response to both abiotic and biotic factors.

2. We inventoried grasshopper communities to document changes in herbivore abundance along

ele-vation gradients and quanti

fied associated shifts in plant palatability. We measured plant palatability

by measuring the growth of Spodoptera littoralis generalist caterpillars fed with the leaves of 172

plant species. We related plant palatability to leaf traits and elevation at the species and community

levels.

3. In congruence with the decrease in grasshopper abundance with elevation, we found that the

mean palatability level of plant communities increases with elevation. In addition, plant palatability

was negatively associated with the community-weighted mean of leaf dry matter content. At the

spe-cies level, plants with high carbon-to-nitrogen ratio were less palatable, while we found no effect of

species mean elevation on plant palatability.

4. Synthesis. Our results suggest that plant communities at higher elevation are composed of species

that are generally more palatable for insect herbivores. Shift in plant palatability with elevation may

thus be the outcome of a relaxation of the in situ herbivore pressure and changes in abiotic conditions.

Key-words: alpine, bioassay, community-weighted mean, grasshoppers, herbivory, plant defence,

plant resistance, plant

–herbivore interactions, Spodoptera littoralis

Introduction

Changes in plant functional properties along environmental

gradients are influenced by both abiotic conditions (e.g.

tem-perature, edaphic factors; e.g. Asner et al. 2014) and biotic

interactions (e.g. plant–animal interactions; Gentry 1988;

Sundqvist, Sanders & Wardle 2013). Along elevation

gradi-ents, the degree of herbivory generally decreases (Reynolds

& Crossley 1997; Garibaldi, Kitzberger & Chaneton 2011;

Metcalfe et al. 2014; Pellissier et al. 2014), concomitantly

with changes in climatic and soil factors (K€orner 2007),

which should influence functional composition of plant

assemblages. For instance, it can be postulated that plants at

higher elevations should invest their limited resources more

in resistance against abiotic stressors (e.g. cold, wind, UV

radiation), other than the unnecessary production of defences

against herbivores (Pellissier et al. 2014). While examples of

intraspecific and interspecific defence relaxation at high

ele-vations or latitude exist (Pennings, Siska & Bertness 2001;

Scheidel & Bruelheide 2001; Salgado & Pennings 2005;

Pel-lissier et al. 2012, 2014; Rasmann et al. 2014a; but see

Moles et al. 2011a,b), it remains unclear whether those

genotypic- or species-speci

fic patterns can be generalized to

entire communities.

*Correspondence author. E-mail: patrice.descombes@wsl.ch

†_{Sharedfirst authorship.} #

Shared last authorship.

http://doc.rero.ch

Published in "Journal of Ecology 105(1): 142–151, 2017"

which should be cited to refer to this work.

Plants have evolved a large diversity of defences to protect

themselves against herbivores (Grime, MacPherson-Stewart &

Dearman 1968; Rhoades 1979; Coley, Bryant & Chapin

1985; Agrawal & Fishbein 2006; Hanley et al. 2007).

Chemi-cal defences primarily rely on carbon- and nitrogen-based

sec-ondary compounds that act as toxins or digestibility reducers

(Mith€ofer & Boland 2012). In complement, physical defences

act via traits that physically decrease the potential for food

acquisition (e.g. leaf toughness, trichomes; Awmack &

Leather 2002; Hanley et al. 2007). The different components

of the plants

’ defence arsenal are expressed together in the

form of syndromes to counter-attack a wide range of

herbi-vore guilds (Agrawal & Fishbein 2006). How elevation

gradi-ents shape the deployment of plant defence syndromes has

not been addressed across many species, partly because of the

high endeavour required for identifying and quantifying

defences of plant species belonging to a wide range of

fami-lies, each one with a unique set of phytochemicals and forms

(Wink 2003). An alternative approach for measuring the net

outcome of plant defence traits is therefore to use bioassays

that quantify the palatability of each plant species against a

unique, but highly polyphagous herbivore (e.g. Grime,

MacPherson-Stewart & Dearman 1968; Edwards, Wratten &

Cox 1985; Pellissier et al. 2012).

Evidence suggests that herbivore communities can in

fluence

plant species composition (e.g. Carson & Root 2000; Fine,

Mesones & Coley 2004) and related plant functional traits.

For instance, Becerra (2007) or Kursar et al. (2009) detected

a signal of chemical overdispersion within communities of

closely related plant species and suggested that plant

–insect

co-evolution modulate plant communities

’ structure. Pellissier

et al. (2013) found a correlation between herbivore and plant

phylogenetic beta diversity, suggesting a link between plant

and insect community assembly, while Richards et al. (2015)

showed that the richness of herbivores correlated with

phyto-chemical diversity in Piper species assemblages. In

accor-dance, we therefore predict that in habitats where the

abundance and richness of herbivores is large, the relative

pressure of distinct herbivore taxa will impose a top-down

control on plant composition and maintain a relatively strong

and diverse defence syndrome (e.g. Kursar & Coley 2003;

Becerra 2007). In contrast, in habitats with lower herbivory

pressure, such as higher elevation, plant species should invest

less in herbivore resistance and plant communities should be

on average more palatable.

Abiotic factors could also shape community-level trait

vari-ation, in turn also modifying plant responses to herbivory

(Rasmann et al. 2014b). For instance, at high elevation, plant

species may produce leaves with increased leaf dry matter

content (LDMC) as an adaptation to severe climatic

condi-tions (K€orner 2003; Dubuis et al. 2013). Because LDMC is

positively related to leaf toughness, where high LDMC plants

are more resistant to physical hazards (i.e. wind, hail;

Cor-nelissen et al. 2003), it might also indirectly confer increased

resistance to insect herbivores (Schuldt et al. 2012; Ibanez

et al. 2013). Therefore, the selective abiotic forces might

pro-mote functional changes in plant communities, which could

indirectly confer more or less resistance to herbivores.

How-ever, the link between herbivore abundance and richness,

abi-otic

conditions

and

plant

palatability

at

the

level

of

communities has not been thoroughly investigated so far.

In this study, we assessed plant palatability for chewing

insect herbivores, physical defence traits and nutritional

com-position of plant species growing between 400 and 3200 m of

elevation, and comprising common plant community

compo-sition at each site. Plant palatability was quanti

fied by

mea-suring the growth of the larvae of the generalist moth

Spodoptera littoralis (Brown & Dewhurst 1975) on leaves

collected in their natural growing locations. The weight of the

larvae should be considered as the integrated measure of the

physical and chemical defence for each plant species, where

high values of larval weight indicate lower defence against

herbivores (Bossdorf et al. 2004; Sch€adler et al. 2007;

Ruhnke et al. 2009; Pellissier et al. 2012). Additionally, we

surveyed for natural grasshopper abundance and richness

along the same elevation gradient to document the expected

decrease in abundance and richness in colder environments.

We chose grasshoppers as indicators of herbivore abundance

since these insects are among the most important herbivores

in calcareous grasslands, where they can remove up to 30%

of the above-ground phanerogam biomass (Blumer & Diemer

1996). Using this data set, we investigated how plant

palata-bility is simultaneously related to leaf functional traits and

elevation, at the species and at the community level, and how

it is likely associated to a shift in herbivore abundance.

Materials and methods

P L A N T A N D O R T H O P T E R A N C O M M U N I T I E S

The study area is located in the Western Swiss Alps (Switzerland, 46°100–46°300N; 6°500–07°100 E) and covers about 700 km2_{, with}

elevations ranging between 375 and 3210 m a.s.l (see Fig. S1 in Sup-porting Information). It is characterized by a temperate climate with annual average temperatures and precipitations varying, respectively, between 8°C and 1200 mm at 600 m, and 5 °C and 2600 mm at 3000 m (Bou€et 1985). The vegetation along the elevation gradient is typical of the calcareous Alps, but also strongly influenced by the human land use and pasture.

In order to sample vegetation data along elevation gradients, a total of 912 plots of 4 m2_{were selected between 400 and 3210 m using a}

random stratified sampling design with regard to elevation, slope and orientation (Hirzel & Guisan 2002), which were inventoried between May and September 2002–2010 (see Fig. S1). All plots were selected among open, non-woody areas by counting with a minimal distance of 200 m between plots in order to limit the potential spatial autocor-relation resulting fromfield inventories performed in the same mea-dow and presenting a very similar plant composition. All vascular plant species present in the plots were inventoried, and their relative abundance estimated using the simplified cover scheme based on Vit-toz & Guisan (2007):<0.1, 0.1–1, 1–5, 5–15, 15–25, 25–50, 50–75 and>75%. The median values of these classes (0.05, 0.5, 3, 10, 20, 37.5, 62.5 and 82.5%) were used in all further analyses.

To assess the abundance of orthopteran along elevation gradients, we randomly selected 175 plots above 1000 m out of the initial 912 plots (see Fig. S1). Between 20th July and 20th September 2012, sites

were visited from low elevation to high elevation by following the spe-cies phenology. Most of the sites below 1800 m a.s.l. (corresponding approximately to the lower limit of the treeline ecotone in the study area; Gehrig-Fasel, Guisan & Zimmermann 2007) were visited a sec-ond time later in the season to collect data on late emerging species due to a longer growing season. The sampling took place between 10 a.m. and 5 p.m. with optimal temperature conditions for insects, and within a 509 50 m area. Most individuals were identified directly on the field to the species level by net catching and/or by their songs. Unknown species and larvae were collected for identification in the lab-oratory and by experts from the Swiss Center from Faunal Cartography (http://www.cscf.ch/). We inventoried a total of 36 orthopteran species, including members of the Caelifera and Ensifera suborder. The abun-dance of each species was then estimated for a 109 10 m area using four classes of abundance: 1–5, 5–20, 20–50, 50–100 individuals. This value of abundance was estimated by means of four 109 10 m sub-plots situated in each cardinal directions (North, South, East and West) at 10 m from the central point and sampled circularly by walking towards the centre. For sites visited a second time later in the season, only the abundance of the additional species was assessed (see Table S1 for the species list and abundance observed on each sampling site). A global measure of abundance for each plot was obtained for Caelifera and Ensifera separately, by summing the median values of the classes of abundance (3, 13, 35 and 75, respectively) of all species. We omitted members of the Tetrigidae family, since these species are principally feeding on algae, lichens, mosses and detritus (e.g. Paran-jape & Bhalerao 1985; Kuravova & Kocarek 2015). We assumed that the measure of global grasshopper abundance is correlated with overall herbivore pressure on the plant community at each site. We extracted elevation information for all sampled plots from a Digital Elevation Model at a resolution of 25 m.

P L A N T P A L A T A B I L I T Y B I O A S S A Y A N D P L A N T T R A I T S

In order to evaluate overall plant palatability for chewing insect herbi-vores, we performed a bioassay experiment on 172 plant species com-monly growing within the study area, using larvae of the African cotton leafworm S. littoralis (Lepidoptera, Noctuidae; Brown & Dewhurst 1975) obtained from Syngenta (Switzerland). We used S. littoralis as a non-adapted species to remove the confounding effect of possible local adaptation to plants. In addition, S. littoralis is a highly generalist insect herbivore, reported to feed on more than 40 families of plants (Brown & Dewhurst 1975), therefore commonly used in similar bioassays (e.g. Edwards, Wratten & Cox 1985; Boss-dorf et al. 2004; Sch€adler et al. 2007; Ruhnke et al. 2009; Pellissier et al. 2012). While herbivores are frequently specialized on a restricted range of host plants due to possible pre-adaptations to quan-titative and qualitative leaf traits of a given plant species (e.g. Mopper 1996; Pellissier et al. 2013; Rasmann et al. 2014b), the assessment of differences in leaf quality by using a generalist herbivore such as S. littoralis should provide a more unbiased measure (Ruhnke et al. 2009). The 172 plant species were selected to represent the diversity of families (number of sampled species relative to total number in each family; Spearman correlation: 0.942) and to represent diversity variation along the elevation gradient (number of sampled versus total species in each elevation bands of 200 m; Spearman correlation: 0.991). Three individuals of each plant species were collected, by selecting plants in sites with contrasting environmental conditions along an elevation gradient transect in the study area (see Fig. S1) so as to cover as much of the total distribution range of each species as possible. Eggs were hatched on wet paper at 20°C without food to ensure a standard size. Once hatched, three larvae were placed on

each individual plant, by placing leaves of each species in distinct Petri dishes for 5 days in a climatic chamber at 24°C (L) and 18 °C (D), 55 5% RH and a 14:10 L:D photoperiod. Completely eaten or dried leaves were replaced during this period with leaves that were stored at 4°C. At the end of the experiment, all the larvae were dried for 72 h at 50°C and weighed. We retained dead larvae into the analyses only if replicated experiments showed the same results (i.e. larvae died in the Petri dishes). Finally, we estimated the average palatability of the 172 plant species by averaging the dry weight of the larvae in and through replicates of the same plant species.

We next measured three traits related to physical leaf defence and leaf nutrient content: specific leaf area (SLA), leaf dry matter content (LDMC) and carbon-to-nitrogen ratio (C:N). SLA and LDMC were estimated for 245 plant species, while C:N was estimated for 251 plant species. We collected 4–20 individuals in sites with contrasting environmental conditions along an elevation gradient transect (see Fig S1) so as to cover as much of the total distribution range of each spe-cies as possible (see Dubuis et al. 2013). Spespe-cies individuals were sampled at the same phenological stage whenever possible by follow-ing the growfollow-ing season accordfollow-ing to altitude, stored in moist bags in a cool box (10°C) and rehydrated previous to measurements by using the partial rehydration method described in Vaieretti et al. (2007). One well-developed entire leaf was then collected per individ-ual (Cornelissen et al. 2003; Perez-Harguindeguy et al. 2013) for trait measurement and dried at 60°C for minimum 4 days. SLA (mm2_mg1_{) was calculated as the area of the leaf divided by its dry}

mass. SLA is correlated with the potential relative growth rate or mass-based photosynthetic rate of a plant, where lower values are associated to higher investments in structural defence strategies, and long leaf lifespan (Cornelissen et al. 2003). LDMC (mg g1) was cal-culated as the ratio of the leaf dry mass to its saturated fresh mass. LDMC is positively related to leaf toughness, where high LDMC plants present higher resistance to physical hazards (i.e. herbivory, wind, hail; Cornelissen et al. 2003) and lower digestibility (Gardarin et al. 2014). Leaf nitrogen (mg g1) and carbon (mg g1) contents were analysed on one sample of mixed ground leaves per species by using an elemental analyser (NC-2500 from CE Instruments). C and N are linked to plant photosynthetic rates and nutrient cycling pro-cesses (Cornelissen et al. 2003). We used the average trait value among all sampled individuals for each species for further analyses.

STAT IST ICA L ANALYS ES

First, we evaluated phylogenetic signal of all plant functional traits described above and plant palatability, by pruning from the published phylogeny of the 231 most frequent and abundant plant species for the study area (Ndiribe et al. 2013), and by calculating Blomberg’s K statistic with the ‘phylosignal’ function as implemented in the ‘ PI-CANTE’ Rpackage (Blomberg, Garland & Ives 2003; Kembel et al.

2010) inR(R Development Core Team 2014,Rversion 3.2.2). Blom-berg’s K statistic compares the observed distribution of the trait val-ues to expectations under a Brownian motion model of trait evolution. K values close to one indicate trait evolution consistent with a Brownian motion model of evolution, while K values close to zero indicate a random distribution of trait values with respect to the phylogeny (Blomberg, Garland & Ives 2003). We tested the signi fi-cance of this test by comparing the observed K value to a null distri-bution generated by comparing 999 randomizations of trait values across the tips of the phylogenetic tree (Kembel et al. 2010).

Next, we related species-level variation in plant palatability to plant trait (SLA, LDMC and C:N) and plant mean elevation, byfitting a phylogenetic least squares model (PGLS) with the package ‘caper’

(Orme et al. 2013) inR, with k transformation for the phylogenetic

tree optimized through maximum likelihood, and by square root trans-forming the response variable and rescaling all variables around their mean. We ensured that the model residuals did not deviate from a normal distribution. PGLS model allows correcting for phylogenetic non-independence among species while correlating two variables. Since collinearity can bias parameter estimation in regression-type models, we calculated a variance inflation factor (VIF; Quinn & Keough 2002) for our predictors by using the‘vifstep’ function in the package‘USDM’ (Naimi 2015). This function calculates a VIF for all

variables and excludes highly correlated variables from the set through a stepwise procedure based on a threshold. An ideal VIF has a value of one. While VIF values higher than 10 are clearly indicative of collinearity issues (Quinn & Keough 2002), it has also been sug-gested that values higher than three could also be indicative of poten-tial collinearity issues (Zuur, Ieno & Elphick 2010; Mundry 2014). We kept all predictors since we found low collinearity (VIF values; SLA= 1.859, C:N = 1.343; LDMC = 1.269, species mean eleva-tion= 1.341). Finally, to ensure that our conclusions were non-sensi-tive to the choice of the elevation metric at the species level, we run additional analysis using elevation range limits with quantile 5% and 95% in addition to the mean elevation.

To investigate the relationships between the plants traits and the environment at the community level, we computed the community-weighted mean (CWM), which represents the mean of a trait for a whole plant community, weighted by the abundance of each species that occur in the community (Garnier et al. 2004). We retained only plots (i.e. 307 plots) whose cover was composed with more than 70% of species for which species trait measurement was available. For each 307 plots, we computed CWM of plant palatability, SLA, LDMC and C:N by using the‘functcomp’ function provided by the package ‘FD’ (Laliberte, Legendre & Shipley 2014). We related CWM of plant palatability to the plant traits and community elevation using an ordi-nary least squares regression model (OLS), by square root transforming the response variable and by rescaling all variables around their mean. We calculated a variance inflation factor (VIF; Quinn & Keough 2002) for our predictors following the same procedure as above. We excluded SLA from the regression model since this variable showed high VIF value (SLA= 4.440; C:N = 2.589; community elevation = 2.263; LDMC= 2.241). After removing SLA, VIF values reached 1.842 for C:N, 1.813 for LDMC and 1.025 for elevation. We also ensured that the model residuals did not deviate from a normal distribution and that the model residuals were not spatially autocorrelated. To ensure that our conclusions were non-sensitive to the choice of cover threshold used for selecting plot (Pakeman & Quested 2007), we run additional analyses when considering only plots whose cover was composed with more than 80% (n= 167) or 90% (n = 72) of species for which species trait measurement was available.

Finally, we related orthopteran (Caelifera and Ensifera suborder separately) richness (i.e. number of species) and abundance (i.e. counts of individuals) to elevation using a linear regression model (i.e. 175 plots). The proportion of variation in Caelifera and Ensifera richness or abundance explained by the model was quantified with the coefficient of determination (R2

).

Results

S P E C I E S -L E V E L A N A L Y S E S

We

found

a

weak

phylogenetic

signal

for

LDMC

(Blomberg’s K: K = 0.335, n = 218, Z-score = 4.981,

P-value

= 0.001),

SLA

(K

= 0.124,

n

= 218,

Z-score

= 2.702, P-value = 0.001), C:N (K = 0.138,

n

= 221, Z-score = 2.217, P-value = 0.001) and plant

palatability

(K

= 0.122,

n

= 133,

Z-score

= 0.627,

P-value

= 0.265; Fig. 1), indicating that the variation of these

plant traits is labile across the phylogeny. Despite general trait

lability, we observed family-level differences in plant

palata-bility (Fig. 1, see Fig. S2). Some plant families such as

Api-aceae, Cyperaceae and Poaceae present lower palatability

levels, while Polygonaceae and Salicaceae exhibited higher

palatability. Other families such as Asteraceae and

Saxifra-gaceae showed high variability (Fig. 1, see Fig. S2).

At the species level and among all predictor variables, only

C:N was signi

ficantly associated with plant palatability in the

PGLS model, which showed a low explained deviance of the

relationship (Table 1a; Fig. 2). We found no relationship

between plant palatability and LDMC, SLA and mean

eleva-tion of sites where the species was found (Table 1a). The

results were consistent, whether we used the mean elevation

at the species level, the quantile 5% or the quantile 95%

representing niche limits (see Table S2).

C O M M U N I T Y - L E V E L A N A L Y S E S

We found that the CWM of plant palatability was signi

fi-cantly associated to the CWM of LDMC and community

ele-vation in OLS multiple regressions (Table 1b, Fig. 3). Plant

communities are more palatable at higher elevation, where

they are composed of plant species affording lower levels of

LDMC (Table 1b, Fig. 3). In contrast to the results at the

Fig. 1. Phylogeny of angiosperms palatability (n = 133). The trait mapped correspond to the mean larval weight (square root trans-formed) of Spodoptera littoralis after 5 days of feeding on leaf plant samples, as a measure of plant palatability. Bigger circles indicate a higher palatability of the plant to generalist chewing insect herbivores (see Fig. S2 for a complete phylogeny with plant species names). [Colourfigure can be viewed at wileyonlinelibrary.com]

species level, the CWM of plant palatability was not

associ-ated to the CWM of C:N in OLS multiple regressions

(Table 1b). We found no autocorrelation in the residuals of

the CWM model (Moran’s I = 0.009; P-value = 0.209).

Overall, we found that the results were not sensitive to the

threshold considered for selecting plot communities, whether

it is 70, 80 or 90% of cover in plots for which species trait

measurement was available (see Table S3).

Finally, orthopteran richness (i.e. number of species) and

abundance (i.e. counts of individuals) were both negatively

correlated to elevation in Caelifera (linear regression;

rich-ness:

n

= 175,

R

= 0.407,

t-value

= 10.970,

slope

= 0.004, P-value <0.001; abundance: n = 175,

R

= 0.212, t-value = 6.925, slope = 0.093, P-value

<0.001; Fig. 4) and Ensifera (linear regression; richness:

n

= 175, R

= 0.528, t-value = 13.99, slope = 0.002,

P-value

<0.001;

abundance:

n

= 175,

R

= 0.257,

t-value

= 7.828, slope = 0.017, P-value <0.001; Fig. 4).

We also found a strong correlation between orthopteran

rich-ness and abundance in Caelifera (Spearman correlation:

q = 0.845, R

_{= 0.714, P-value <0.001) and Ensifera}

(Spear-man correlation:

q = 0.823, R

= 0.678, P-value <0.001), as

a result of the contrast between low elevation rich and

abun-dant orthopteran communities versus high elevation poor

communities (Fig. 4).

Discussion

S P E C I E S - L E V E L P L A N T P A L A T A B I L I T Y A L O N G E L E V A T I O N G R A D I E N T S

Plant defence levels may vary along an elevation gradient by

following two expectations. The

first one relies on the fact

that producing defensive chemicals is costly (Gulmon &

Mooney 1986; Gershenzon 1994; Cipollini, Purrington &

Bergelson 2003) and predicts a decrease of defence with

ele-vation, in line with the concomitant decrease in herbivore

abundance. The second one relies on the resource availability

hypothesis (Coley, Bryant & Chapin 1985; Endara & Coley

2011), which states that plants with slow growth rates

occur-ring in environments with low resources should be highly

defended against herbivory because of the high cost of tissue

loss in resource-poor environments. Accordingly, as

environ-mental harshness and low resources availability limit plant

growth rate at high elevation, we would expect high elevation

plants to exhibit high levels of defence against herbivory. The

fact that we found no effect of elevation on plant palatability

at the species level (see similar results in Rasmann et al.

2014b) suggests that different species may show dissimilar

sensitivities to herbivore abundance and abiotic conditions,

Table 1. (a) Relationships between plant palatability and four predic-tor variables at the plant species level (n= 129) estimated from phy-logenetic least squares model (PGLS) including all predictor variables and bivariate linear regressions. (b) Relationships between commu-nity-weighted mean (CWM) of plant palatability and four predictor variables at the plant community level estimated from ordinary least squares multiple regressions (OLS) and bivariate linear regressions. CWM was estimated for communities whose plant cover was com-posed with more than 70% of species for which species trait measure-ment was available (n= 307). SLA was not considered in the OLS model since it was highly correlated to the other predictors. The table (a and b) shows the coefficient of determination (R2), the t-value and the standardized regression coefficients (Estimate)

(a) Species level

PGLS Bivariate model

Estimate t-value Estimate t-value R2

SLA 0.009 0.081 0.097 1.093 0.002 LDMC 0.090 0.698 0.178 2.037 0.024* C:N 0.256 2.536* 0.201 2.312 0.033* ELEV 0.062 0.650 0.009 0.101 0.008 R2 0.054* Lambda 0.416 (b) Community level

OLS Bivariate model

Estimate t-value Estimate t-value R2

SLA 0.028 0.489 0.002

LDMC 0.397 6.132*** 0.429 8.298 0.182*** C:N 0.058 0.893 0.279 5.079 0.075*** ELEV 0.343 7.034*** 0.327 6.042 0.104*** R2 _0.292***

SLA, specific leaf area; LDMC, leaf dry matter content; C:N, carbon-to-nitrogen content; ELEV, mean elevation.

*P < 0.05, ***P < 0.001.

Fig. 2. Partial regression plot (i.e. added variable plot) calculated from PGLS model (n= 129) showing the independent contribution of C:N in explaining plant species palatability variation (i.e. solid black line; Table 1a) at the species level. The axis represents the residuals of the models (x-axis: C:N~ SLA + LDMC + ELEV; y-axis: Palata-bility~SLA + LDMC + ELEV). Before fitting the PGLS model, plant palatability was square root transformed and all variables were rescaled around their mean. The dashed line corresponds to the mean of the y-axis. SLA (specific leaf area); LDMC (leaf dry matter con-tent); C:N (carbon-to-nitrogen concon-tent); ELEV (mean elevation). Plants are more palatable when their leaves are more nutrient rich (i.e. low C:N).

leading to a lack of a clear trend when considering all species

individually (Rasmann et al. 2014b).

In accordance with the common rule of insects being

mostly deficient in nitrogen, larvae performed slightly better

on plants with a low carbon-to-nitrogen content (Table 1a,

Fig. 2; White 1984; Elser et al. 2000). Other leaf variables

such as leaf toughness (represented here by LDMC and SLA)

were not associated to plant palatability, which is in contrast

with previous results in which SLA was associated with

investment in physical leaf defence (Coley 1983; Cornelissen

et al. 2003; but see Louda & Rodman 1996). Since values of

SLA and LDMC are highly plastic across sites, it is likely

that more samples would be required to capture intraspeci

fic

variability and to accurately catch the multifaceted dimensions

of plant defences based on physical traits only (Ackerly 2009;

Cornwell & Ackerly 2009). In addition, this study did not

take into account plant secondary metabolites specifically,

which are known to play a preponderant role in driving insect

performance (Fraenckel 1959; but see Carmona, Lajeunesse

& Johnson 2011). Plants chemical defences are difficult to

study across such a large plant taxonomic scale because the

secondary

metabolites

involved

are

extremely

diverse

(Mith€ofer & Boland 2012; Kant et al. 2015), but may largely

explain the observed difference in palatability among plant

species.

We found a weak phylogenetic signal for plant leaf traits

such as SLA, LDMC and C:N, even if the latter were

signifi-cantly different from a random distribution of plant traits

across the phylogeny. This suggests that variation of several

plant functional traits across species shows a pattern of

adap-tation to the environment, with limited phylogenetic inertia

(Wiens & Graham 2005; Losos 2008). Similarly, we found

no phylogenetic signal for plant palatability suggesting that

mechanisms of plant defence (physical and chemical

com-bined) are probably species-specific, and independent of the

phylogeny at the scale we performed our analysis. Indeed,

palatability could show phylogenetic conservatism at deeper

(e.g. family level) nodes of the phylogeny. For instance, most

of the species in the Apiaceae, Poaceae and Cyperaceae show

lower palatability to insect herbivores (Fig. 1), suggesting that

Fig. 3. Partial regression plots (i.e. added variable plots) calculated from OLS model showing the independent contribution of (a) LDMC and (b) community elevation in explaining plant community palatability variation (i.e. solid black line; Table 1b). The axis represents the residuals of the models. CWM of plant palatability was estimated for communities whose plant cover was composed with more than 70% of species for which species trait measurement was available (n= 307). Before fitting the OLS model, plant palatability was square root transformed and all variables were rescaled around their mean. The grey area corresponds to the 95% confidence interval around the mean. The dashed line corresponds to the mean of the y-axis. LDMC (leaf dry matter content); C:N (carbon-to-nitrogen content); ELEV (mean elevation). Communities are more palatable at high elevation sites and/or when they are composed of plant species presenting lower LDMC.

Fig. 4. Changes in (a) orthopteran richness measured as the number of species and (b) orthopteran abundance (i.e. counts of individuals) along the elevation gradient for Caelifera (black dots) and Ensifera (grey triangles) suborder. Each dot and each triangle represent a sampled community where orthopteran abundance and richness were estimated (n= 175; see Fig. S1). The grey and black areas correspond to the 95% confidence interval around the mean for Ensifera and Caelifera, respectively. Orthopteran Caelifera and Ensifera are more abundant and diverse on low elevation sites.

family-specific defence traits, such as secondary metabolites

(e.g. furanocoumaris for Apiaceae; Berenbaum, Zangerl &

Nitao 1986), or silica content in grasses and sedges

(O’Rea-gain & Mentis 1989; Vicari & Bazely 1993; Massey, Ennos

& Hartley 2006; Massey & Hartley 2009) might drive

conser-vatism of plant–herbivore interaction (Futuyma & Agrawal

2009). However, testing for family differences would require

a larger species sampling than considered here.

C OM MU N I T Y - L E V E L P L A N T P A L A T A B I L I T Y A L O N G E L E V A T I O N G R A D I E N T S

At the community level, we observed an increase of the

over-all plant palatability with elevation, suggesting that the most

dominant species in plant communities growing at higher

ele-vations are generally more palatable to herbivores than their

low elevation counterparts. This result is in line with the

observed decrease of orthopteran abundance and richness with

elevation and suggests that dominant plants respond to

reduced herbivore abundance by relaxing their defences

over-all (Pellissier et al. 2012), supporting the hypothesis of

defence relaxation with elevation (Gulmon & Mooney 1986;

Gershenzon 1994). Previous studies showed a similar

relation-ship between plant chemical and biomechanical defence traits

at the community level and herbivore abundance and richness

(Becerra 2007; Peeters, Sanson & Read 2007; Richards et al.

2015). However, those study at the level of communities

never extended at higher latitude or elevation.

Together with elevation, we found that the mean

nity-level value of LDMC was also related to plant

commu-nity palatability (Table 1b, Fig. 3). In other words, plant

communities situated at higher elevation and/or presenting

higher mean community-level value of LDMC are generally

less palatable to herbivores. This result parallels

finding at the

species level in which leaf toughness was associated with

investment in physical leaf defence (Coley 1983; Choong

1996; Hochuli 2001; Cornelissen et al. 2003; Clissold et al.

2009; Ibanez et al. 2013; but see Louda & Rodman 1996).

Abiotic factors could also shape community-level trait

varia-tion, in turn also modifying plant responses to herbivory

(Rasmann et al. 2014b). For instance, plant species at high

elevation may

first produce leaves with increased leaf dry

matter content (LDMC) as an adaptation to severe climatic

conditions (K€orner 2003; Dubuis et al. 2013), thus increasing

resistance to physical hazards (i.e. wind, hail; Cornelissen

et al. 2003). In turn, this might indirectly confer increased

resistance to arthropod herbivores (Schuldt et al. 2012; Ibanez

et al. 2013).

In sum, we found contrasted results in the species- and

community-level analyses. Plant palatability was related to

LDMC and elevation at the community level, while only C:N

showed a trend in species-based analyses driven by a few

species (Fig. 2). While in species-level analyses each species

is given the same weight, at the community level, trait values

are weighed by the dominance of species (i.e. higher weight

to dominant plant species; Garnier et al. 2004; Pellissier et al.

2012). This suggests that the relationships between plant

palatability, LDMC and elevation are driven predominantly

by species with a higher cover. Dominant plant species are

expected to be more frequently targeted by herbivores and to

invest more energy in physical defence to generalist herbivore

(Pellissier et al. 2015). For plant species predominantly

rely-ing on physical defences, a decrease in LDMC would directly

result in higher palatability. In contrast, for species also

rely-ing on chemical defences, the relationship would be less

clear. Moreover, the increase in plant palatability at higher

elevation communities might suggest that dominant plants

better modulate defences to shifting ecological conditions,

such as biotic interaction with herbivores. Together, our

results indicate that plant dominance should be included when

addressing the ecology and evolution of plant defence

strate-gies.

Our

findings indicate that the measure of community-level

plant palatability could be one of the processes explaining

how climate change will affect plant community composition

in the future. Many insects are currently limited in their

distri-bution by temperature and length of the growing season

(Maclean 1983; Strathdee et al. 1993; Whittaker & Tribe

1996; Miles, Bale & Hodkinson 1997; Bird & Hodkinson

1999; Hodkinson et al. 1999; Ritchie 2000). However, the

higher temperatures associated with climate change will allow

invertebrate herbivores to track climatic changes (Wilson

et al. 2007) to an extent that plants cannot (Grabherr,

Got-tfried & Pauli 1994 but see Cannone, Sgorbati & Guglielmin

2007). For instance, B€assler et al. (2013) found an upslope

shift of the upper range margin for insects that exceeded

expectations based on climatic changes. The predicted future

increase in herbivore abundance at high elevations under

cli-mate changes (Rasmann et al. 2014b) will increase the

pres-sure on palatable plant communities. Herbivory could have in

turn a disproportionate negative impact on the cover of these

species (Brown & Gange 1989) and may cause severe shifts

in the pattern of plant and soil carbon cycling (Metcalfe et al.

2014). Ultimately, palatable species could disappear from

high elevation communities because insects may selectively

feed on them, facilitating their replacement by lower elevation

plant species (Rasmann et al. 2014b). Moreover, increasing

stress conditions through climate change (e.g. drought) could

also alter the defence of plants against insect herbivory by

reducing investments in secondary metabolites (Gutbrodt,

Mody & Dorn 2011).

In conclusion, our study showed that the decrease in plant

palatability at higher elevation can be scaled up to entire

com-munities. In contrast, we found only weak trends when

look-ing at species individually. Future work uslook-ing

community-wide metabolomics approaches for obtaining more

informa-tion on leaf chemistry, and identifying the dominant chemical

defence strategies along the elevation gradient might help

overcoming these obstacles (Van Dam & Poppy 2008; Jansen

et al. 2009). High elevation areas represent refuges from

her-bivory for some alpine plants: they are excluded from low

elevations because of the high herbivory pressure in lowland

(Galen 1990; Bruelheide & Scheidel 1999; Bruelheide 2003).

However, climate change is expected to lift current climate

barriers and herbivore colonization at higher elevation may

promote fast plant communities turnover, in which less

palat-able high elevation plants will be selected.

Acknowledgements

We thank all the people who helped with field work, Oliver Kindler and Roland Reist (Syngenta, Stein, Switzerland) for providing S. littoralis eggs, and the editor and two anonymous referees who provided valuable comments to improve the manuscript. The project wasfinanced by the Swiss National Fund grant_{‘Lif3web’ number 31003A-162604 to L.P.}

Data accessibility

Data from this paper can be accessed through figshare: http://dx.doi.org/ 10.6084/m9._{figshare.3803955 (Descombes et al. 2016).}

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Supporting Information

Additional Supporting Information may be found in the online version of this article:

Figure S1. Location of the study area in the western Alps of Switzer-land.

Figure S2. Phylogeny of angiosperms palatability. Table S1. Species list observed on the sampling sites. Table S2. Results of PGLS models at the species level. Table S3. Results of OLS models at the community level.